viruses Review Current Understanding of the Molecular Basis of Venezuelan Equine Encephalitis Virus Pathogenesis and Vaccine Development Anuj Sharma * and Barbara Knollmann-Ritschel Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA; [email protected] * Correspondence: [email protected] Received: 27 December 2018; Accepted: 7 February 2019; Published: 18 February 2019 Abstract: Venezuelan equine encephalitis virus (VEEV) is an alphavirus in the family Togaviridae. VEEV is highly infectious in aerosol form and a known bio-warfare agent that can cause severe encephalitis in humans. Periodic outbreaks of VEEV occur predominantly in Central and South America. Increased interest in VEEV has resulted in a more thorough understanding of the pathogenesis of this disease. Inflammation plays a paradoxical role of antiviral response as well as development of lethal encephalitis through an interplay between the host and viral factors that dictate virus replication. VEEV has efficient replication machinery that adapts to overcome deleterious mutations in the viral genome or improve interactions with host factors. In the last few decades there has been ongoing development of various VEEV vaccine candidates addressing the shortcomings of the current investigational new drugs or approved vaccines. We review the current understanding of the molecular basis of VEEV pathogenesis and discuss various types of vaccine candidates. Keywords: Venezuelan equine encephalitis virus; alphavirus; inflammation; encephalitis; viral and host factors; blood brain barrier; pathogenesis; vaccines 1. Introduction Venezuelan equine encephalitis virus (VEEV) is a member of genus Alphavirus in the family Togaviridae. VEEV complex is a group of 14 antigenic varieties divided into 7 species. The VEEV species include four antigenic varieties namely IA/B, IC, ID, and IE, all of which cause human disease that is indistinguishable between the antigenic varieties [1]. Subtypes IA/B and C are epizootic strains that cause fulminant disease and high mortality in equines. Subtypes ID and IE are enzootic strains that are typically avirulent in equines; however, IE can be neurovirulent in equines. VEEV is an enveloped virus which is maintained in nature in a cycle between rodents and mosquitoes with epizootic strains sporadically causing outbreaks in equines and humans (Figure1)[ 2,3]. The geographic distribution and outbreaks of VEEV in equines and humans has been reviewed in detail by Aguilar et al. [1] and Weaver et al. [4]. VEEV is a Category B agent as defined by the Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety level 3 containment is required for handling of live virulent strains of VEEV. Two live-attenuated strains of VEEV, namely TC-83 and V3526, can be safely handled at biosafety level 2 containment [5]. VEEV infection in humans starts with an asymptomatic incubation period of 1–5 days followed by the onset of a febrile illness characterized by fever, headache, nausea, vomiting, myalgia, ocular pain, lower back pain and diarrhea lasting for 1–4 days [6]. The short febrile illness may progress into fulminant encephalitis causing convulsions, hemiparesis, behavioral changes, and alteration of consciousness. A more severe infection can occur which is associated with hemichorea, seizures, and stupor or coma [7–9]. Mortality in humans is <1%, Viruses 2019, 11, 164; doi:10.3390/v11020164 www.mdpi.com/journal/viruses Viruses 2018, 10, x 2 of 32 illness characterized by fever, headache, nausea, vomiting, myalgia, ocular pain, lower back pain and diarrhea lasting for 1-4 days [6]. The short febrile illness may progress into fulminant encephalitis causingViruses 2019 convulsions,, 11, 164 hemiparesis, behavioral changes, and alteration of consciousness. A more2 of 32 severe infection can occur which is associated with hemichorea, seizures, and stupor or coma [7–9]. Mortality in humans is <1%, but the incidence of neurological disease can be up to 14% in infected patientsbut the incidence[10]. The ofmouse neurological is the most disease common can be model up to 14%used in to infected investigate patients VEEV [10 ].pathogenesis The mouse isas the it closelymost common mimics modelthe biphasic used to investigatecourse of peripheral VEEV pathogenesis replication as itfollowed closely mimicsby infection the biphasic of the course central of nervousperipheral system replication (CNS) followed as seen in by severe infection cases of theof human central VEEV nervous infection system (CNS)i.e., the as initial seen infebrile severe illness cases dueof human to virus VEEV replication infection in the i.e., peripheral the initial febrileorgans illness followed due by to virusa second replication phase of in CNS the peripheralinfection (Figure organs 2)followed [11]. In byhealthy a second immunocompetent phase of CNS infection adult mice (Figure models2)[ 11 ].such In healthy as CD-1 immunocompetent Swiss [12], Balb/c adult[13], miceand C57BL6models [14] such mice, as CD-1 infection Swiss with [12], wild-type Balb/c [13 VEEV], and causes C57BL6 a biphasic [14] mice, disease infection similar with to wild-typethe severe VEEVform ofcauses disease a biphasic in humans. disease VEEV similar can tobe thedetected severe in form local of lymph disease nodes in humans. as early VEEV as 6 canh post be detected infection. in Animalslocal lymph become nodes viremic as early within as 6 12 h posth of infection. By Animals 12 h post become infection, viremic VEEV within can 12also h be of infection.detected inBy other 12 h peripheral post infection, organs. VEEV The can virus also replicates be detected in the in otherlymphoid peripheral tissue organs.e.g., lymph The nodes virus replicatesand spleen, in asthe well lymphoid as in non-lymphoid tissue e.g., lymph organs nodes including and spleen, the heart, as well lung, as kidney, in non-lymphoid and pancreas. organs In the including lymphoid the tissues,heart, lung, VEEV kidney, induces and cellular pancreas. necrosis In the and lymphoid an inflammatory tissues, VEEV cell inducesresponse. cellular Loss or necrosis alteration and of an germinalinflammatory center cell structures response. in Lossthe sp orleen alteration is observed of germinal as early centeras 24 h structures post infection in the and spleen is accompanied is observed byas lymphocyte early as 24 h karryohrexis post infection and and apoptosis, is accompanied as well by as lymphocyte macrophage karryohrexis infiltration. andRecovery apoptosis, starts as by well 72 has post macrophage infection. infiltration.The virus is Recovery cleared from starts peripheral by 72 h post organs infection. within The 4–5 virus days isof cleared infection. from In peripheralthe brain, VEEVorgans first within appears 4–5 in days the ofolfactory infection. lobe In around the brain, 36–48 VEEV h post first infection. appears The in virus the olfactory then spreads lobe rapidly around throughout36–48 h post the infection. brain. ThePerivascular virus then cuffing spreads and rapidly lymphocyte throughout infiltration the brain. are Perivascular observed cuffing72 h post and infection.lymphocyte Viral infiltration spread are and observed corresponding 72 h post infection. inflammation Viral spread are characterized and corresponding by inflammationperivascular lymphocyticare characterized cuffing, by perivascular gliosis, neurodegeneration, lymphocytic cuffing, andgliosis, vacuolization neurodegeneration, of neuropil, and which vacuolization increase in of intensityneuropil, with which time. increase The kinetics in intensity of viral with spread time. Theinto kinetics the brain of is viral dependent spread into on the brainroute isof dependentinfection. Viruson the appears route of in infection. the CNS Virus much appears earlier in when the CNS infection much earlier is via whenaerosol infection exposure is via due aerosol to the exposure direct infectiondue to the of direct olfactory infection neuroepithelium of olfactory neuroepithelium by aerosolized of by VEEV aerosolized particles, of VEEVcompared particles, to a subcutaneous compared to a infectionsubcutaneous route infection which requires route which virus requires replication virus inreplication lymphoid intissue lymphoid and the tissue development and the development of viremia inof order viremia for inthe order virusfor to then thevirus be able to to then infect be ablethe olfactory to infect neuroepitheliu the olfactory neuroepitheliumm [13,15,16]. Additionally, [13,15,16]. extensiveAdditionally, hepatocellular extensive degeneration hepatocellular and degeneration interstitial pneumonia and interstitial are seen pneumonia in human are VEEV seen infections; in human however,VEEV infections; these are however, not the primary these are pathological not the primary findings pathological in animal findings models inused animal for investigating models used VEEVfor investigating infection [17]. VEEV Characterization infection [17]. Characterizationof VEEV pathogenesis of VEEV has pathogenesis been aided by has the been availability aided by theof clearlyavailability defined of clearly mutant defined strains mutant developed strains by developed point mutations by point in mutations the full-length in the clone full-length of wild-type clone of Trinidadwild-type Donkey Trinidad (TrD) Donkey strain (TrD) (subtype strain (subtype I/AB) of I/AB) VEEV of (Table VEEV (Table1), which1), which based based on the on theextent extent of attenuationof